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What is RNA-seq?

RNA-seq (RNA sequencing) is a powerful and widely used technique for analysing the quantity and sequences of RNA in a sample. It provides a snapshot of the entire transcriptome—the complete set of RNA transcripts produced by the genome at a given time.

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In practice, both pipelines are highly regarded, and the best choice often comes down to the specific requirements of your research project and the resources available.

Overview of pipeline tools:

source: https://nf-co.re/rnaseq/3.14.0

1. Merge re-sequenced FastQ files (cat)

2. Auto-infer strandedness by subsampling and pseudoalignment (fq, Salmon)

3. Read QC (FastQC)

4. UMI extraction (UMI-tools)

5. Adapter and quality trimming (Trim Galore!)

6. Removal of genome contaminants (BBSplit)

7. Removal of ribosomal RNA (SortMeRNA)

8. Choice of multiple alignment and quantification routes:

   1. STAR -> Salmon

   2. STAR -> RSEM

   3. HiSAT2 -> NO QUANTIFICATION

9. Sort and index alignments (SAMtools)

10. UMI-based deduplication (UMI-tools)

11. Duplicate read marking (picard MarkDuplicates)

12. Transcript assembly and quantification (StringTie)

13. Create bigWig coverage files (BEDTools, bedGraphToBigWig)

14. Extensive quality control:

    1. RSeQC

    2. Qualimap

    3. dupRadar

    4. Preseq

    5. DESeq2

    6. Kraken2 -> Bracken on unaligned sequences; optional

15. Pseudoalignment and quantification (Salmon or ‘Kallisto’; optional)

16. Present QC for raw read, alignment, gene biotype, sample similarity, and strand-specificity checks (MultiQC, R)